Growth of Coliphage T7 in Salmonella typhimurium

A mutant of Salmonella typhimurium was found to be sensitive to killing by coliphage T7 because of an alteration in its surface properties. However, the infections were abortive and studies with (32)P-labeled T7 grown in Escherichia coli B (T7.B) indicated that the phage DNA was restricted by S. typhimurium. When a mutant T7 which survived the restriction and produced plaques on Salmonella (T7.S) was passed through one cycle of growth in E. coli B, its ability to grow in Salmonella was lost, indicating that host-controlled restriction and modification are operative in this system. Restrictionless S. typhimurium mutants were isolated that permit the growth of not only T7.S but also T7.B and coliphage T3. The physiology of T7 production in the restrictionless host is nearly identical to that in Escherichia coli.     

Lysozymes from bacteriophages T3 and T5.

Lysozymes produced in host cells infected with bacteriophages T3 and T5 were found to have the same enzymatic specificity toward the peptidoglycan from Escherichia coli as T7 phage lysozyme, which has been shown to be an N-acetylmuramyl-L-alanine amidase.     

Different restriction of bacteriophages T3 and T7 by P1-lysogenic cells and the role of the T3-coded SAMase.

The intracellular growth of the phages T3 and T7 is restricted in the presence of the Escherichia coli prophage P1. Phage T3 has a higher ability to express its genome and to damage the host cell than T7. This partial protection of T3 against P1 restriction is due to the T3-coded SAMase, an enzyme which degrades S-adenosylmethionine, the cofactor of the P1 restriction endonuclease. Since we did not observe DNA cleavage in vivo, we conclude that the in vivo action of the P1 nuclease is limited to a SAM-dependent repressor-like binding to T3 and T7 DNA, while further reactions with the DNA (modification vs cleavage) are blocked.     

A T3 and T7 recombinant phage acquires efficient adsorption and a broader host range

It is usually thought that bacteriophage T7 is female specific, while phage T3 can propagate on male and female Escherichia coli. We found that the growth patterns of phages T7M and T3 do not match the above characteristics, instead showing strain dependent male exclusion. Furthermore, a T3/7 hybrid phage exhibits a broader host range relative to that of T3, T7, as well as T7M, and is able to overcome the male exclusion. The T7M sequence closely resembles that of T3. T3/7 is essentially T3 based, but a DNA fragment containing part of the tail fiber gene 17 is replaced by the T7 sequence. T3 displays inferior adsorption to strains tested herein compared to T7. The T3 and T7 recombinant phage carries altered tail fibers and acquires better adsorption efficiency than T3. How phages T3 and T7 recombine was previously unclear. This study is the first to show that recombination can occur accurately within only 8 base-pair homology, where four-way junction structures are identified. Genomic recombination models based on endonuclease I cleavages at equivalent and nonequivalent sites followed by strand annealing are proposed. Retention of pseudo-palindromes can increase recombination frequency for reviving under stress.     

Abortive Infection of F-Plasmid-Containing Escherichia coli Cells by Bacterial Virus T7 Is Determined by the Right End of T7 Gene 1

Phage T7 infects male (F-plasmid-carrying) Escherichia coli cells abortively, whereas the closely related phage T3 grows normally. The inability or ability of phage to replicate in male host cells depends on whether the right end of gene 1 (coding for the phage-specific RNA polymerase) consists of T7 or T3 DNA base sequences.     

Changes in the nature of the sensitivity of an E. coli M strain carrying Inc-group R plasmids, to coliphages T3 and T7

After the transfer of prototype plasmids R6K (IncX), R387 (IncK), R27 (IncH1) and T (IncN) to E. coli M nalr the appearance of histidine-dependent mutants (R27, T), histidine-leucine-dependent mutants (R6K), methionine-proline-dependent mutants (R387) was observed among the resulting transconjugates. The mutations of E. coli M nalr R+ cells induced by the introduction of the plasmids were accompanied by the transformation of the cells from the S-form into the R-form. In contrast to the prototrophs E. coli M nalr, the auxotrophs carrying plasmids R6K, R27, T acquired sensitivity to phage T7, and the methionine-proline-dependent mutant became sensitive to phages T and T7. The above-mentioned plasmids rendered E. coli M cells capable of synthetizing the donor pili. But the adsorption of phages T3 and T7 on the auxotrophic cells, both with and without plasmids, occurred due to their interaction with the cell-wall receptors.     

T3 and T7 Bacteriophage Deoxyribonucleic Acid-Directed Enzyme Synthesis In Vitro

The T3 phage enzymes S-adenosyl methionine cleaving enzyme and lysozyme and the T7 lysozyme were synthesized in a deoxyribonucleic acid (DNA)-dependent, cell-free system derived from uninfected Escherichia coli. The data presented suggest that these enzymes are encoded in that portion of the DNA which is transcribed early after infection.     

The effect of the T7 and Escherichia coli DNA-binding proteins at the replication fork of bacteriophage T7

In this paper we compare the effect of single-stranded DNA-binding proteins of bacteriophage T7 (gene 2.5 protein) and of Escherichia coli (SSB) at the T7 replication fork. The T7 gene 4 protein acts processively as helicase to promote leading strand synthesis and distributively as primase to initiate lagging strand synthesis by T7 DNA polymerase. On a nicked double-stranded template, the formation of a replication fork requires partial strand displacement so that gene 4 protein may bind to the displaced strand and unwind the helix catalytically. Both the T7 gene 2.5 protein and E. coli SSB act stoichiometrically to promote this initial strand displacement step. Once initiated, processive leading strand synthesis is not greatly stimulated by the single-stranded DNA-binding proteins. However, the T7 gene 2.5 protein, but not E. coli SSB, increases the frequency of initiation of lagging strand synthesis by greater than 10-fold. The results suggest a specific interaction of the T7 gene 2.5 protein with the T7 replication apparatus.     

Genetic analysis of gene 1.2 of bacteriophage T7: isolation of a mutant of Escherichia coli unable to support the growth of T7 gene 1.2 mutants.

The product of gene 1.2 of bacteriophage T7 is not required for the growth of T7 in wild-type Escherichia coli since deletion mutants lacking the entire gene 1.2 grow normally (Studier et al., J. Mol. Biol. 135:917-937, 1979). By using a T7 strain lacking gene 1.2, we have isolated a mutant of E. coli that was unable to support the growth of both point and deletion mutants defective in gene 1.2. The mutation, optA1, was located at approximately 3.6 min on the E. coli linkage map in the interval between dapD and tonA; optA1 was 92% cotransducible with dapD. By using the optA1 mutant, we have isolated six gene 1.2 point mutants of T7, all of which mapped between positions 15 and 16 on the T7 genetic map. These mutations have also been characterized by DNA sequence analysis, E. coli optA1 cells infected with T7 gene 1.2 mutants were defective in T7 DNA replication; early RNA and protein synthesis proceeded normally. The defect in T7 DNA replication is manifested by a premature cessation of DNA synthesis and degradation of the newly synthesized DNA. The defect was not observed in E. coli opt+ cells infected with T7 gene 1.2 mutants or in E. coli optA1 cells infected with wild-type T7 phage.     

Differences in sensitivity of T3, T7, T4 and lambda phages to bleomycin and phleomycin]

In contrast to phage lambda the phages T3, T7 and T4 are not inhibited by as much as 150 microgram bleomycin/ml, while the chemically related antibiotic phleomycin increasingly inhibits the propagation of the phages in the order T4-T3-lambda. 20 microgram phleomycin/ml inhibit T3 by 95%. The resistance against bleomycin is surprising, because 10 microgram BM/ml block completely the colony-forming capacity of the host bacterium. The drug resistance of the phage growth correlates with the weak decrease of phage DNA synthesis, while the host cell DNA synthesis ceases rapidly. In accordance with these data is the in vivo inhibition of Escherichia coli cells and the in vitro degradation of their DNA. However, a contradiction exists between the in vivo resistance of T3 and T4 and the in vitro susceptibility of their DNA against nucleolytical fragmentation by bleomycin. The mechanism of the insensitivity of T3, T7 and T4 against bleomycin is unknown.     

Effect of temperature on RNA synthesis in Escherichia coli CRT 266 (dna Bts) following infection with bacteriophage T3]

Infection of the temperature-sensitive E. coli CRT 266 (dnaBts) with T3-phages at the temperature of 30 degrees C and 35 degrees C, respectively, induced T3-specific RNA synthesis with a maximum rate at 7 min (30 degrees C) and 4.5 min (35 degrees C) after infection. At temperatures above 40 degrees C no T3-induced RNA synthesis could be observed. Infection of E. coli CR 34--45 (dnaB+) with T3 phages at 30 degrees C, 35 degrees C and at temperatures above 40 degrees C, however, produced T3-specific RNA synthesis. The maximum of T3-induced RNA synthesis could be observed between 7 min and 3 min depending on the temperature during infection. The inability to form T3-specific RNA after infection of E. coli CRT 266 at nonpermissive temperatures may be a cause for the absence of the formation of T3 phages and lysis of the host cells.     

T7 and E. coli share homology for replication-related gene products

Recently, the complete nucleotide sequence of the bacteriophage T7 genome was determined and 50 genes were identified on the genome. We compared amino acid sequences of all the gene products of T7 and replication-related gene products of E. coli. As a result, we found that T7 and E. coli share homology for each pair of exonuclease, DNA primase and helix-destabilizing protein. For E. coli, these gene products are known to be involved in the process of discontinuous DNA replication. These observations suggest that T7 and E. coli have a common origin for a part of their replication systems.     

Interactions of gene 2.5 protein and DNA polymerase of bacteriophage T7.

Bacteriophage T7 gene 2.5 protein has been shown to interact with T7 DNA polymerase (the complex of T7 gene 5 protein and Escherichia coli thioredoxin) by affinity chromatography and fluorescence emission anisotropy. T7 DNA polymerase binds specifically to a resin coupled to gene 2.5 protein and elutes from the resin when the ionic strength of the buffer is raised to 250 mM NaCl. In contrast, T7 gene 5 protein alone binds more weakly to gene 2.5 protein, eluting when the ionic strength of the buffer is 50 mM NaCl. Thioredoxin does not bind to gene 2.5 protein. Steady-state fluorescence emission anisotropy gives a dissociation constant of 1.1 +/- 0.2 microM for the complex of gene 2.5 protein and T7 DNA polymerase, with a ratio of gene 2.5 protein to T7 DNA polymerase in the complex of 1:1. Nanosecond emission anisotropic analysis suggests that the complex contains one monomer each of gene 2.5 protein, gene 5 protein, and thioredoxin. The ability of T7 gene 2.5 protein to stimulate the activity and processivity of T7 DNA polymerase is compared with the ability of three other single-stranded DNA-binding proteins: E. coli single-stranded DNA-binding protein, T4 gene 32 protein, and E. coli recA protein. All except E. coli recA protein stimulate the activity and processivity of T7 DNA polymerase; E. coli recA protein inhibits these activities.     

Amber Mutants of Bacteriophages T3 and T7 Defective in Phage-directed Deoxyribonucleic Acid Synthesis

Amber mutants of the related phages T3 and T7 were isolated and tested for their ability to restore-as the wild type does-thymidine incorporation in ultraviolet (UV)-irradiated, UV-sensitive, nonpermissive host bacteria (Escherichia coli B(s-1)). Most amber mutants had this ability. However, in both T3 and T7, mutants unable to promote thymidine incorporation under these conditions were found and classified into two well-defined complementation groups: T3DO-A and T3DO-B, T7DO-A and T7DO-B. Infection of B(s-1) cells with representatives of groups DO-A had the following characteristics: (i) phage-directed uridine uptake in UV-irradiated cells was reduced to less than 20% of normal; (ii) breakdown of host deoxyribonucleic acid (DNA) was delayed and incomplete; (iii) no serum-blocking antigens appeared; (iv) no cell lysis occurred; (v) the ability to exclude the heterologous wild type was impaired. Amber mutants of the DO-B groups, infecting B(s-1), were able to: (i) promote an efficient phage-directed uridine uptake in UV-irradiated cells; (ii) bring about rapid breakdown of host DNA; (iii) synthesize serum-blocking antigens; (iv) lyse the host cells, generally after the normal latent period; (v) exclude efficiently the heterologous wild type. Although physiological similarities between the respective DO-A mutants or DO-B mutants of T3 and T7 were evident, no physiological cross-complementation occurred, and genetic crosses gave no evidence of genetic homologies between groups of T3 and T7.     

Coiled Rings of DNA Released from Cells Infected with Bacteriophages T7 or T4 or from Uninfected Escherichia coli

The replicating intracellular DNA of phage T7 was labeled at high specific activity with tritiated thymidine. The DNA of uninfected Escherichia coli was similarly labeled. Portions of cells which contained replicating phage T7 or E. coli DNA were lysed by a lysozyme, freeze-thaw, sodium lauryl sulfate procedure, and the DNA was spread on Millipore membranes for visualization by autoradiography. The DNA of phage T7 appeared to be highly concatenated reaching lengths of up to 721 mum. Much of the DNA of phage T7 and E. coli was retained in compact globular structures. In addition, orderly coiled rings of varying diameter up to about 43 mum were regularly observed. Similar coiled ring structures were also observed in autoradiographs of replicating phage T4 DNA which had been prepared in previous experiments. Worcel and Burgi (27) have presented evidence that E. coli chromosomes, when gently extracted from cells, are in a multilooped and superhelically twisted configuration. The coiled rings which we have observed may correspond to the relaxed, multilooped configurations which they find when the superhelical twists have been relieved by one or more nicks in each loop.     

One step engineering of T7-expression strains for protein production: increasing the host-range of the T7-expression system

The T7-expression system has been very useful for protein expression in Escherichia coli. However, it is often desirable to over-express proteins in species other than E. coli. Here, we constructed an inducible broad-host-range T7-expression transposon, which allows simple one-step construction of T7-expression strains in various species, providing the option to over-express proteins of interest in a broader host-range. This transposon contains the T7 RNA polymerase driven by the lacUV5 promoter, which is repressed by the lac-repressor. Leaky expression is prevented by the presence of T7-lysozyme on this construct. The complete T7-expression system is flanked by mariner transposon repeats of the suicidal R6Kgammaori plasmid, pBT20-Deltabla. Stable integration of the whole system is possible by a one-step selection for a Flp-excisable Gm(R)-marker. We showed the engineering of E. coli, Pseudomonas aeruginosa, Erwinia carotovora, Salmonella choleraesuis, Agrobacterium tumefaciens, and Chromobacterium violaceum strains with this construct and demonstrated the expression of the Burkholderia pseudomallei Asd protein in these hosts, by induction with isopropyl-beta-d-thiogalactopyranoside (IPTG).     

利用重组噬菌体诱导表达的大肠杆菌表达系统

将编码噬菌体T7RNA聚合酶的基因克隆至噬菌体M13mpl8RFDNA中,置于lac启动子的控制之下,得到了可表达T7 RNA聚合酶的重组噬菌体M13HEP。利用该噬菌体感染含T7启动子表达质粒的宿主菌以提供T7RNA聚合酶,可以诱导T7启动子控制下的外源基因的表达。该噬茵体诱导表达系统已成功地表达了多种外源基因,特别是一些表达产物对宿主菌有毒性的基因。同时,通过细菌接合将F',因子从大脑杆菌XL1-blue转至大肠杆菌HMS174,构建了新的大脑杆菌菌株HMSl74F,,使得T7表达质粒构建、表达及单链制备可以在同一菌株中完成,得到了一个完整的T7表达系统。